Many genera of bacteria assemble layers composed of repetitive, regularly aligned, proteinaceous sub-units on the outer surface of the cell. These layers are essentially two-dimensional paracrystalline arrays, and being the outer molecular layer of the organism, directly interface with the environment. Such layers are commonly known as S-layers and are found on members of every taxonomic group of walled bacteria including: Archaebacteria; Chlamydia; Cyanobacteria; Acinetobacter; Bacillus; Aquaspirillum; Caulobacter; Clostridium; Chromatium. Typically, an S-layer will be composed of an intricate, geometric array of at least one major protein having a repetitive regular structure. In many cases, such as in Caulobacter, the S-layer protein is synthesized by the cell in large quantities and the S-layer completely envelopes the cell and thus appears to be a protective layer.
Caulobacter are natural inhabitants of most soil and freshwater environments and may persist in waste water treatment systems and effluents. The bacteria alternate between a stalked cell that is attached to a surface, and an adhesive motile dispersal cell that searches to find a new surface upon which to stick and convert to a stalked cell. The bacteria attach tenaciously to nearly all surfaces and do so without producing the extracellular enzymes or polysaccharide “slimes” that are characteristic of most other surface attached bacteria. They have simple requirements for growth. The organism is ubiquitous in the environment and has been isolated from oligotrophic to mesotrophic situations. Caulobacters are known for their ability to tolerate low nutrient level stresses, for example, low phosphate levels. This nutrient can be limiting in many leachate waste streams, especially those with high levels of iron or calcium.
Freshwater Caulobacter producing S-layers may be readily detected by negative stain transmission electron microscopy techniques. Caulobacter may be isolated using the methods outlined by MacRae, J. D. and Smit (1991) Applied and Environmental Microbiology 57:751-758, which take advantage of the fact that Caulobacter can tolerate periods of starvation while other soil and water bacteria may not and that they all produce a distinctive stalk structure, visible by light microscopy (using either phase contrast or standard dye staining methods). Once Caulobacter strains are isolated in a typical procedure, colonies pended 2% ammonium molybdate negative stain and applied to plastic-filmed, carbon-stabilized 300 or 400 mesh copper or nickel grids and examined in a transmission electron microscope at 60 kilovolt accelerating voltage, as described in Smit, J. (1986) “Protein Surface Layers of Bacteria”, in Outer Membranes as Model System, M. Inouge, Ed. J. Wiley & Sons, at page 343-376. S-layers are seen a two-dimensional geometric patterns most readily on those cells in a colony that have lysed and released their internal contents.
The S-layer of different freshwater Caulobacter is hexagonally arranged with a similar centre—centre dimension and antisera raised against the S-layer protein of C. crescentus strain CB15 reacts with S-layer proteins from other Caulobacter (see: Walker, S. G., et al. (1992) (J. Bacteriol. 174:1783-1792). All S-layer proteins isolated from Caulobacter may be substantially purified using the same extraction method (pH extraction). All strains appear to have a lipopolysaccharide (LPS) reactive with antisera against the CB15 strain lipopolysaccharide species. The LPS appears to be required for S-layer attachment.
The S-layer elaborated by freshwater isolates of Caulobacter are visibly indistinguishable from the S-layer produced by Caulobacter crescentus strains CB2 and CB15. The S-layer proteins from the latter strains have approximately 100.000 m.w. although sizes of S-layer proteins from other species and strains will vary. The protein has been characterized both structurally and chemically. It is composed of ring-like structures spaced at 22 nm intervals arranged in a hexagonal manner on the outer membrane. The S-layer is bound to the bacterial surface and may be removed by low pH treatment or by treatment with a calcium chelator such as EDTA.
The similarity of S-layer proteins in different strains of Caulobacter permits the use of a cloned S-layer protein gene of one Caulobacter strain for retrieval of the corresponding gene in other Caulobacter strains (see: Walker, S. G. et al. (1992) [supra]: and, MacRae, J. D. et al. (1991) [supra].
Expression, secretion and optionally, presentation of a heterologous polypeptide in Caulobacter provides advantages not previously seen in systems using organisms such as E. coli and Salmonella in which fusion products using different surface proteins have been reported. All known Caulobacter strains are believed to be harmless and are nearly ubiquitous in aquatic environments. In contrast, many Salmonella and E. coli strains are pathogens. Consequently, expression and secretion of a heterologous polypeptide using Caulobacter as a vehicle will have the advantage that the expression system will be stable in a variety of outdoor environments and may not present problems associated with the use of a pathogenic organism. Furthermore, Caulobacter are natural biofilm forming species and may be adapted for use in fixed biofilm bioreactors. The quantity of S-layer protein that is synthesized and is secreted by Caulobacter is high, reaching 12% of the cell protein. The unique characteristics of the repetitive, two-dimensional S-layer would also make such bacteria ideal for use as an expression system, or as a presentation surface for heterologous polypeptides. This is desirable in a live vaccine to maximize presentation of the antigen or antigenic epitope. In addition, use of such a presentation surface to achieve maximal exposure of a desired polypeptide to the environment results in such bacteria being particularly suited for use in bioreactors or as carriers for the polypeptide in aqueous or terrestrial outdoor environments.
The invention described in the PCT application published Sep. 18, 1997 under WO 97/34000 describes the C-terminal region of Caulobacter crescents S-layer protein as being essential for secretion of S-layer protein in that species. Heterologous polypeptides may be conveniently expressed and secreted by a host Caulobacter when the polypeptide is expressed as a fusion with the C-terminal secretion signal. Further studies with C. crescentus have demonstrated that the species employs a type I secretion system which involves an uncleaved C-terminal secretion signal on the surface layer protein (RsaA) and several transport proteins encoded by genes 3′ to the surface layer protein gene (rsaA) (Amram, P. and Smit, J. (1998) Journal of Bacteriology 180:3062-3069).
A typical type I secretion system uses three transport protein components. One such component, the ABC transporter, is embedded in the inner membrane, contains an ATP-binding region, recognizes the C-terminal secretion signal of the substrate protein, and hydrolyzes ATP during the transport process. Another component, the membrane fusion protein (MFP) is anchored in the inner membrane and appears to span the periplasm. The remaining component is an outer membrane protein (OMP) that is thought to interact with the MFP to form a channel that extends from the cytoplasm through the two membranes to the outside of the cell. In C. crescentus, the ABC transporter and the MFP proteins have been termed RsaD and RsaE (respectively) and their genes are immediately 3′ of rsaA. Further downstream is the rsaF gene which is believed to encode the OMP.
It is desirable to provide for the use of Caulobacter species other than C. crescentus in the expression and secretion of heterologous polypeptides from a host organism.